The long-term goal of this project is to determine the role of human cytomegalovirus (HCMV) in the acceleration of vascular diseases such as atherosclerosis, restenosis, and transplant vascular sclerosis (TVS). HCMV is associated with TVS and CR in solid organ transplant patients and we have observed that RCMV significantly accelerates the development of TVS and CR in heart allografts in the rat transplant model. Although we can detect the presence of virus in the transplanted heart throughout the disease course, the number of infected cells in the allograft does not account for the global effects of RCMV infection of the organ in the development of TVS. This lack of correlation suggests that infected cells are influencing the microenvironment via paracrine mechanisms. Analysis of allografts with RCMV-accelerated TVS for host cell gene expression by microarray analysis revealed a significant number of genes involved in tissue remodeling (WH) and angiogenesis (AG) that were highly up-regulated in the organ transplant. In addition our preliminary results indicate that virus-free and serum-free cellular supernatants obtained from HCMV- but not mock- infected cells induce both WH & AG in in vitro assays. Mass Spec analysis of the HCMV secretome identified multiple angiogenic and WH agonists and antagonists that most likely contribute to the tissue remodeling processes. We hypothesize that the spectrum of cytokines and growth factors secreted by CMV-infected cells significantly contribute to the acceleration of TVS and CR in heart allografts through the stimulation of WH and AG. Therefore, in this project we propose the use of viral genetics to unravel the WH and AG mechanisms involved in CMV acceleration of TVS and CR. In the first specific aim of this project we will use HCMV WT and mutant viruses as tools in combination with gel-free LC Mass Spec proteomics and in vitro WH and AG assays to identify specific viral genes and cellular proteins as well as the mechanisms through which viral genes induce tissue-remodeling events. In the second aim of this project we will knock-out the HCMV correlate gene(s) in RCMV and test this virus(es) in WH and AG assays, as well as analyze the viral secretome(s) by Mass Spec analysis. Lastly, we will examine the effect of these mutations on the ability of the virus to accelerate TVS in the RCMV rat heart transplant model. The anticipation is that multiple HCMV genes are responsible for the induction of WH and AG and that we will be able to separate some of these processes to examine the effect of mutation of correlate RCMV genes in the acceleration of TVS.